湍流剪切力在血管内皮细胞激活整合素alpha5-YAP通路的机制及其意义

Atherosclerotic lesions often happen in bifurcations and curved regions of artery vessel, which suggests that local variations in hemodynamic forces play a significant role in atherogenesis. However, the molecular mechanism is still unclear. Our previous study demonstrated that disturbed flow induced translocation of integrin α5 (α5) to the cell membrane lipid rafts and activated, an important effector of Hippo pathway, YAP was also activated in vascular endothelial cells, but their relationship has not yet been elucidated. The purpose of this study is to explore the molecular mechanism of disturbed flow activation of α5-YAP cascade and its role in atherogenesis. We hypothesize that disturbed flow promotes α5 lipid raft translocation, and be activated via an interaction with GPCR, in turn, activates YAP through RhoA and/or tyrosine kinase pathway(s), thus regulates the transcription of downstream genes, leading to endothelial activation. With disturbed flow activation of α5-YAP cascade as a central, using proteomics, biochemistry, cell and molecular biology tools and genetically engineered mice, we will systematically investigate 1) the molecular mechanism of disturbed flow induced α5 lipid raft transposition and activation in endothelial cells; 2) the mechanism of downstream pathway activation via the interaction between α5 and GPCR; 3) the molecular mechanism of disturbed flow promotes α5-induced YAP activation, and 4) α5-YAP pathway as targets, drug screening and verification of small molecule compounds to provide theoretical and experimental evidence for early intervention of atherogenesis.

动脉粥样硬化常见于发生湍流的动脉血管分叉或弯曲处，其分子机制不清。前期研究表明在血管内皮细胞湍流促使整合素α5（α5）向细胞膜脂筏转位并激活；同时活化Hippo通路的效应分子YAP，激活内皮，但二者关系尚未阐明。本研究旨在揭示湍流激活α5-YAP通路的机制及其在动脉粥样硬化发生中的作用。提出假说：湍流促使α5脂筏内转位，通过与GPCR相互作用而激活；通过RhoA通路和/或氨酸激酶活化YAP，进而调控下游基因转录，导致内皮的激活。拟将以湍流激活α5-YAP通路为中心，运用组学、生化、细胞及分子生物学手段和基因工程小鼠，重点研究在内皮细胞中：1）洄流引起α5脂筏转位并激活的分子机制；2）α5与GPCR相互作用对下游通路激活的机制；3）湍流诱导α5促进YAP活化的分子机制及其意义；4）以α5-YAP通路为靶点进行小分子化合物的筛选验证。以期为干预动脉粥样硬化早期病变的药物筛选提供理论及实验依据。

湍流剪切力介导的内皮功能失调是动脉粥样硬化性发生的始动因素，本项目的主要目标在于阐明α5-YAP通路在这一过程中的关键作用和分子调控机制。项目执行以来，围绕研究目标以及拟解决的关键科学问题，按原计划5个主要研究内容全面完成了研究工作：1）湍流引起内皮细胞中α5脂筏转位并激活的分子机制：我们的研究表明Piezo1-PTP1B-Annexin A2 信号通路介导湍流诱导的α5的转位以及血管内皮激活。2）GPCR与α5相互作用对α5脂筏聚集及其下游通路激活的影响：我们发现细胞外基质蛋白COMP通过与α5的相互作用抑制α5激活的调控机制，揭示了基质蛋白COMP的内源性保护作用。同时，阐明了COMP对血压的保护作用机制，为动脉粥样硬化和高血压的治疗提供了新思路和新靶点。3）血管内皮细胞中激活的α5促进YAP入核的分子机制：我们研究揭示了α5/c-Abl/YAP信号通路在内皮激活的新机制，并发现c-Abl抑制剂bosutinib在低剂量时对动脉粥样硬化起保护作用，大剂量时引起血压升高的分子机制。4）YAP激活在湍流引起的内皮激活中的地位及分子机制：为了探讨Hippo/YAP通路在内皮激活中的作用，我们通过蛋白组学分析及免疫共沉淀等技术发现了在血管内皮中与YAP蛋白结合的新的转录因子STAT3。5）以α5-YAP为靶点的小分子化合物筛选及在动物模型的验证：我们发现了天然小分子化合物Harmine通过稳定PTPN14促进YAPY357的去磷酸化，使其不能向细胞核聚集进而激活，从而改善内皮功能障碍。项目支持下在 Circ Res、J Clin Invest、Hypertension、Br J Pharmacol、Signal Transduct Target Ther等高水平期刊发表论文。共发表 SCI 论文 10 篇，其中 IF≥10的 5 篇，平均IF =15.9。授权国家发明专利2项。